Bacillus calmette-guerin (bcg) and antigen presenting cells for treatment of bladder cancer

ABSTRACT

Combination therapeutics for the treatment of cancer include the use of immune effector cells, IL-15 based superagonists and one or more immunotherapeutic agents such as Bacillus Calmette-Guerin (BCG).

FIELD OF THE INVENTION

Embodiments of the invention are directed to treatment of bladder cancerusing compositions that induce an immune response to bladder cancercells. In particular, the compositions include antigen presenting cells,isolated from subjects treated with Bacillus Calmette-Guerin (BCG) andcultured with IL-15 based superagonists.

BACKGROUND

Urothelial carcinoma is the most common bladder cancer (BC) with anincidence of approximately 90%. At diagnosis two-third of urothelialcarcinomas are non-muscle invasive and one-third are muscle invasive.Non-muscle invasive bladder cancer (NMIBC) is characterized by a highrisk of recurrence (30-85% depending on stage and grade) aftertransurethral resection of the bladder tumor (TURBT). Moreover, up to17% of all NMIBC will eventually progress to muscle invasive bladdercancer (MIBC). Non-muscle invasive bladder cancer (NMIBC) begins andstays in the cells lining the bladder without growing into the deepermain muscle layer of the bladder, and accounts for the majority (70-80%)of patients diagnosed with bladder cancer (stages Ta, T1, or CIS).Approximately 30% of patients present with muscle-invasive disease(stages T2-T4). Bladder cancer has the highest recurrence rate of anymalignancy.

A significant limitation of current Bacillus Calmette-Guerin (BCG)treatment is the lack of response in a substantial number of patients.For example, depending on the endpoint studied, up to 50% of patientsfail to respond and show progression of the cancer to muscle invasivedisease. Additionally, approximately one-third of patients thatinitially respond to therapy will show tumor recurrence (Sylvester, R.J., Int J Urol., February; 18(2):113-20 (2011)). Patients withrecurrence after BCG therapy may present with urothelial carcinoma ofthe upper urinary tract or the prostatic urethra, which is notaccessible to BCG. Also, patients with no measurable pre-existing T-cellimmunity to BCG (due to previous BCG immunization or natural exposure tomycobacteria) have a lower recurrence-free survival rate (Biot, C., etal., Sci Transl Med., June 6; 4(137):137ra72 (2012)). Patients withBCG-unresponsive non-muscle-invasive bladder cancer (NMIBC) have limitedtreatment options and the standard of care for these patients is radicalcystectomy.

SUMMARY

Embodiments of the invention are directed to the treatment of bladdercancer utilizing, in particular aspects, antigen presenting cells, suchas, for example, dendritic cells isolated from patients who have beenadministered an immunotherapeutic, such as Bacillus Calmette-Guerin(BCG). The antigen presenting cells are cultured ex vivo with aninterleukin-15 (IL-15) based superagonist prior to administering to thesubject. In certain embodiments, the dendritic cells are isolated fromsubjects who have not been treated with BCG.

In certain embodiments, a method for treating cancer comprisesadministering to the subject an effective dose of an immunotherapeuticagent; isolating an immune effector cell from the biological sample ofthe subject; culturing the immune effector cells with a compositioncomprising an IL-15:IL-15Rα fusion protein complex such as anIL-15N72D:IL-15RαSu/Fc complex, and reinfusing the cultured immuneeffector cells to the subject. In certain embodiments, theimmunotherapeutic is BCG. In certain embodiments, the cultured immuneeffector cells are administered in combination with BCG and/or an IL-15based superagonist and/or a chemotherapeutic agent.

In certain embodiments, a method for treating cancer comprises isolatingan immune effector cell from a biological sample of the subject;culturing the immune effector cells with BCG and a compositioncomprising an IL-15:IL-15Rα fusion protein complex such as anIL-15N72D:IL-15RαSu/Fc complex, and reinfusing the cultured immuneeffector cells to the subject. In certain embodiments, the culturedimmune effector cells are administered in combination with BCG and/or anIL-15 based superagonist and/or a chemotherapeutic agent.

In certain embodiments, a method of inducing an anti-bladder cancerimmune response in a subject in need thereof, comprises administering tothe subject an effective dose of Bacillus Calmette-Guerin (BCG);isolating dendritic cells from a urine sample of the subject; culturingthe dendritic cells with a composition comprising an IL-15:IL-15Rαfusion protein complex such as an IL-15N72D:IL-15RαSu/Fc complex,reinfusing the dendritic cells to the subject, thereby inducing ananti-bladder cancer immune response.

In certain embodiments, a vaccine composition comprises BacillusCalmette-Guerin (BCG) primed dendritic cells and an IL-15:IL-15Rα fusionprotein complex such as an IL-15N72D:IL-15RαSu/Fc complex. In certainembodiments, the dendritic cells are isolated from a urine sample of asubject diagnosed with urothelial/bladder carcinoma. In certainembodiments, the vaccine composition comprises an adjuvant.

In certain embodiments, the IL-15 based superagonist comprises a dimericIL-15RαSu/Fc and two IL-15N72D molecules. In certain aspects of thesoluble fusion protein complexes of the invention, the IL-15 polypeptideis an IL-15 variant having a different amino acid sequence than nativeIL-15 polypeptide. The human IL-15 polypeptide is referred to herein ashuIL-15, hIL-15, huIL15, hIL15, IL-15 wild type (wt), and variantsthereof are referred to using the native amino acid, its position in themature sequence and the variant amino acid. For example, huIL15N72Drefers to human IL-15 comprising a substitution of N to D at position72. In one aspect, the IL-15 variant functions as an IL-15 agonist asdemonstrated, e.g., by increased binding activity for the IL-15RβγCreceptors compared to the native IL-15 polypeptide. Alternatively, theIL-15 variant functions as an IL-15 antagonist as demonstrated by e.g.,decreased binding activity for the IL-15RβγC receptors compared to thenative IL-15 polypeptide.

In these and other embodiments, the immune effector cell comprises Tcells, B cells, natural killer (NK) cells, natural killer T (NK-T)cells, dendritic cells, mast cells, myeloid-derived phagocytes orcombinations thereof. In certain embodiments, the immune effector cellis a dendritic cell. In these and other embodiments, the immune effectorcells comprising: autologous, allogeneic, haplotype matched, haplotypemismatched, haplo-identical, xenogeneic cells or combinations thereof.

In certain embodiments, the neoplastic disease or cancer compriseswherein the wherein cancer comprises bladder cancer, a glioblastoma,prostate cancer, hematological cancer, B-cell neoplasms, multiplemyeloma, B-cell lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia, acute myeloid leukemia, cutaneous T-cell lymphoma, T-celllymphoma, a solid tumor, urothelial/bladder carcinoma, melanoma, lungcancer, renal cell carcinoma, breast cancer, gastric and esophagealcancer, head and neck cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, B cell non-Hodgkin lymphoma, and squamouscell head or neck carcinoma. In certain embodiments, the cancer isurothelial/bladder carcinoma.

In certain embodiments, the methods further comprise administering oneor more chemotherapeutic agents.

In certain embodiments, a pharmaceutical composition comprises atherapeutically effective amount of an immunotherapeutic agent, and/oran IL-15N72D:IL-15RαSu/Fc complex (N-803). In certain embodiments theimmunotherapeutic agent comprises Bacillus Calmette-Guerin (BCG) and theN-803 comprises a dimeric IL-15RαSu/Fc and two IL-15N72D molecules.

In certain embodiments, the treatment approaches of the invention couldalso be combined with any of the following therapies: radiation,chemotherapy, surgery, therapeutic antibodies, immunomodulatory agents,proteasome inhibitors, pan-DAC inhibitors, H-DAC inhibitors, checkpointinhibitors, adoptive cell therapies include CAR T and NK cell therapyand vaccines.

Cell therapies of the invention comprise administration of an effectiveamount of immune effector cells. For example, an effective amount ofdendritic cells is between 1×10⁴ cells/kg and 1×10¹⁰ cells/kg, e.g.,1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, and1×10¹² cells/kg, or such amounts that can be isolated by leukapheresis.Alternatively, expanded immune cells are administered as a fixed dose orbased on body surface area (i.e., per m²). Cells can be administeredafter ex vivo expansion or cryogenically preserved and administeredafter thawing (and washing as needed). The adoptively transferred immuneeffector cells or pharmaceutical compositions embodied herein, isadministered at least one time per month, e.g., twice per month, onceper week, twice per week, once per day, twice per day, every 8 hours,every 4 hours, every 2 hours, or every hour. Suitable modes ofadministration for the adoptively transferred immune cells includesystemic administration, intravenous administration, or localadministration. Suitable modes of administration for the pharmaceuticalcomposition include systemic administration, intravenous administration,local administration, subcutaneous administration, intramuscularadministration, intratumoral administration, inhalation, andintraperitoneal administration.

In certain embodiments, the dendritic cells are loaded with a tumorantigen, e.g. urothelial carcinoma antigens.

In some embodiments of the invention, the patient is pretreated orpreconditioned to facilitate engraftment or survival of the adoptivelytransferred cells. Examples of preconditioning include treatment withcyclophosphamide and fludarabine. Additionally, the patient may betreated with agents that promote activation, survival or persistence ofthe adoptively transferred cells pre- and/or post-cell transfer.

Exemplary effective doses of the IL-15:IL-15Rα complex (N-803) includebetween 0.1 μg/kg and 100 mg/kg body weight, e.g., 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, or 900 μg/kg body weight or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg body weight.

In some cases, the N-803 and/or the BCG are administered daily, e.g.,every 24 hours, or, continuously or several times per day, e.g., every 1hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours,every 11 hours, or every 12 hours.

Exemplary effective daily doses of N-803 include between 0.1 μg/kg and100 μg/kg body weight, e.g., 0.1, 0.3, 0.5, 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 μg/kg bodyweight.

Alternatively, the N-803 is administered about once per week, e.g.,about once every 7 days. Or, the N-803 is administered twice per week,three times per week, four times per week, five times per week, sixtimes per week, or seven times per week. Exemplary effective weeklydoses of N-803 include between 0.0001 mg/kg and 4 mg/kg body weight,e.g., 0.001, 0.003, 0.005, 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, or 4mg/kg body weight. For example, an effective weekly dose of N-803 isbetween 0.1 μg/kg body weight and 400 μg/kg body weight. Alternatively,N-803 is administered at a fixed dose or based on body surface area(i.e., per m²).

In some cases, subjects receive two 6-week cycles consisting of 4 weeklyN-803 intravenous doses followed by a 2-week rest period. Ultimately,the attending physician or veterinarian decides the appropriate amountand dosage regimen.

In certain embodiments, a kit for the treatment of bladder cancercomprises an effective amount of immune effector cells, animmunotherapeutic agent, and/or an IL-15N72D:IL-15RαSu/Fc complex(N-803), and directions for the treatment of bladder cancer. In certainembodiments the immunotherapeutic agent comprises BacillusCalmette-Guerin (BCG) and the N-803 comprises a dimeric IL-15RαSu/Fc andtwo IL-15N72D molecules.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant a peptide, nucleic acid molecule, small compound,nucleic acid based moiety, antibody, antibody-based molecule, protein,protein-based molecule and/or substance for use in the prevention,treatment, management and/or diagnosis of cancer.

By “N-803” is meant a complex comprising IL-15N72D noncovalentlyassociated with a dimeric IL-15RαSu/Fc fusion protein and having immunestimulating activity. In one embodiment, the IL-15N72D and/orIL-15RαSu/Fc fusion protein comprises one, two, three, four or moreamino acid variations relative to a reference sequence. An exemplaryIL-15N72D amino acid sequence is provided below.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

The invention includes antibodies or fragments of such antibodies, solong as they exhibit the desired biological activity. Also included inthe invention are chimeric antibodies, such as humanized antibodies.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. Humanization can beperformed, for example, using methods described in the art, bysubstituting at least a portion of a rodent complementarity-determiningregion for the corresponding regions of a human antibody.

The term “antibody” or “immunoglobulin” is intended to encompass bothpolyclonal and monoclonal antibodies. The preferred antibody is amonoclonal antibody reactive with the antigen. The term “antibody” isalso intended to encompass mixtures of more than one antibody reactivewith the antigen (e.g., a cocktail of different types of monoclonalantibodies reactive with the antigen). The term “antibody” is furtherintended to encompass whole antibodies, biologically functionalfragments thereof, single-chain antibodies, and genetically alteredantibodies such as chimeric antibodies comprising portions from morethan one species, bifunctional antibodies, antibody conjugates,humanized and human antibodies. Biologically functional antibodyfragments, which can also be used, are those peptide fragments derivedfrom an antibody that are sufficient for binding to the antigen.“Antibody” as used herein is meant to include the entire antibody aswell as any antibody fragments (e.g. F(ab′)2, Fab′, Fab, Fv) capable ofbinding the epitope, antigen or antigenic fragment of interest.

By “binding to” a molecule is meant having a physicochemical affinityfor that molecule.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeErlotinib (TARCEVA™, Genentech/OSI Pharm.), Bortezomib (VELCADE™,Millennium Pharm.), Fulvestrant (FASLODEX™, Astrazeneca), Sutent(SU11248, Pfizer), Letrozole (FEMARA™, Novartis), Imatinib mesylate(GLEEVEC™, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin(Eloxatin, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin(Sirolimus, RAPAMUNE™, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline),Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), andGefitinib (IRESSA™, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen),alkylating agents such as Thiotepa and CYTOXAN™ cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozcicsin,carzcicsin and bizcicsin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin yl and calicheamicin omega 1 (Angew Chem. Intl.Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, anthramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, strcptonigrin, strcptozocin, tubcrcidin,ubenimcx, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacytidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosinc;arabinoside (“Ara-C”); cyclophosphamidc; thiotcpa; taxoids, e.g., TAXOL™paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR™ gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX™(tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON™ (toremifene); (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate),AROMASIN™ (exemestane), formestanie, fadrozole, RIVISOR™ (vorozole),FEMARA™ (letrozole), and ARIMIDEX™ (anastrozole); (iii) anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);(iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipidkinase inhibitors; (vii) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, such as, for example, PKC-alpha, Ralf andH-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g.,ANGIOZYME™ (ribozyme)) and a HER2 expression inhibitor; (ix) vaccinessuch as gene therapy vaccines, for example, ALLOVECTIN™ vaccine,LEUVECTIN™ vaccine, and VAXID™ vaccine; PROLEUKIN™ rIL-2; LURTOTECAN™topoisomerase 1 inhibitor; ABARELIX™ rmRH; (x) anti-angiogenic agentssuch as bevacizumab (AVASTIN™, Genentech); and (xi) pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include neoplasias.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component, alone or in a combination, to providethe desired effect. For example, by “an effective amount” is meant anamount of a compound, alone or in a combination, required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. For example, a fragment maycontain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 nucleotides or amino acids. However, theinvention also comprises polypeptides and nucleic acid fragments, solong as they exhibit the desired biological activity of the full lengthpolypeptides and nucleic acid, respectively. A nucleic acid fragment ofalmost any length is employed. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length (including all intermediate lengths) are included inmany implementations of this invention. Similarly, a polypeptidefragment of almost any length is employed. For example, illustrativepolypeptide segments with total lengths of about 10,000, about 5,000,about 3,000, about 2,000, about 1,000, about 5,000, about 1,000, about500, about 200, about 100, or about 50 amino acids in length (includingall intermediate lengths) are included in many implementations of thisinvention.

As used herein, the term “IL-15:IL-15Rα fusion protein complex” is acomplex having IL-15 non-covalently or covalently bound to IL-15Rα.IL-15Rα can be either soluble or membrane bound. In some embodiments,IL-15Rα is the soluble domain of the native IL-15Rα polypeptide. Thesoluble IL-15Rα can be the IL-15Rα sushi domain or IL-15RαΔE3. In somecases, the soluble IL-15Rα is covalently linked to a biologically activepolypeptide and/or to an IgG Fc domain. The IL-15 can be either IL-15 orIL-15 covalently linked to a second biologically active polypeptide. Insome cases, IL-15 is covalently bound to the IL-15Rα domain via alinker. The IL-15 can also represent an IL-15 variant comprises one,two, three, four or more amino acid variations relative to a referencesequence. In one embodiment the IL-15 is IL-15N72D. In anotherembodiment, the IL-15:IL-15Rα fusion protein complex is N-803.

As used herein, the term “immune cells” generally includes white bloodcells (leukocytes) which are derived from hematopoietic stem cells (HSC)produced in the bone marrow “Immune cells” includes, e.g., lymphocytes(T cells, B cells, natural killer (NK) cells) and myeloid-derived cells(neutrophil, eosinophil, basophil, monocyte, macrophage, dendriticcells).

The term “immune effector cell,” as used herein, refers to a cell thatis involved in an immune response, e.g., in the promotion of an immuneeffector response. Examples of immune effector cells include T cells,e.g., alpha/beta T cells and gamma/delta T cells, B cells, naturalkiller (NK) cells, natural killer T (NK-T) cells, dendritic cells, mastcells, and myeloid-derived phagocytes. “Immune effector function orimmune effector response,” as that term is used herein, refers tofunction or response, e.g., of an immune effector cell, that enhances orpromotes an immune attack of a target cell. For example, an immuneeffector function or response refers a property of a T or NK cell thatpromotes killing or the inhibition of growth or proliferation, of atarget cell. In the case of a T cell, primary stimulation andco-stimulation are examples of immune effector function or response.

As used herein, the term “in combination” in the context of theadministration of a therapy to a subject refers to the use of more thanone therapy for therapeutic benefit. The term “in combination” in thecontext of the administration can also refer to the prophylactic use ofa therapy to a subject when used with at least one additional therapy.The use of the term “in combination” does not restrict the order inwhich the therapies (e.g., a first and second therapy) are administeredto a subject. A therapy can be administered prior to (e.g., 1 minute, 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second therapy to a subject which had, has, or issusceptible to cancer. The therapies are administered to a subject in asequence and within a time interval such that the therapies can acttogether. In a particular embodiment, the therapies are administered toa subject in a sequence and within a time interval such that theyprovide an increased benefit than if they were administered otherwise.Any additional therapy can be administered in any order with the otheradditional therapy.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. An “isolated cell”e.g. a dendritic cell can be isolated from other cell populations, byfor example, biomarkers. See, for example, Nair, S., Archer, G. E., &Tedder, T. F. (2012). Isolation and generation of human dendritic cells.Current protocols in immunology, Chapter 7, Unit7.32.doi:10.1002/0471142735.im0732s99. Kits for isolating dendritic cells canbe obtained from, STEMCELL Technologies Inc. Cambridge, Mass.

A “purified” or “biologically pure” protein is sufficiently free ofother materials such that any impurities do not materially affect thebiological properties of the protein or cause other adverseconsequences. That is, a nucleic acid or peptide of this invention ispurified if it is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Purity and homogeneity are typically determined using analyticalchemistry techniques, for example, polyacrylamide gel electrophoresis orhigh performance liquid chromatography. The term “purified” can denotethat a nucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. For a protein that can be subjected tomodifications, for example, phosphorylation or glycosylation, differentmodifications may give rise to different isolated proteins, which can beseparately purified.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “neoplasia” is meant a disease or disorder characterized by excessproliferation or reduced apoptosis. Illustrative neoplasms for which theinvention can be used include, but are not limited to bladder cancer,leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myeloblastic leukemia, acute promyelocyticleukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, gastric and esophagealcancer, head and neck cancer, rectal cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, glioblastomamultiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Inparticular embodiments, the neoplasia is multiple myeloma, beta-celllymphoma, urothelial/bladder carcinoma or melanoma.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

By “reduces” is meant a negative alteration of at least 5%, 10%, 25%,50%, 75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout ortherebetween.

By “specifically binds” is meant a compound or antibody that recognizesand binds a polypeptide of the invention, but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.The subject is preferably a mammal in need of such treatment, e.g., asubject that has been diagnosed with B cell lymphoma or a predispositionthereto. The mammal is any mammal, e.g., a human, a primate, a mouse, arat, a dog, a cat, a horse, as well as livestock or animals grown forfood consumption, e.g., cattle, sheep, pigs, chickens, and goats. In apreferred embodiment, the mammal is a human.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. It will be appreciated that,although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated.

Treatment of patients with neoplasia may include any of the following:Adjuvant therapy (also called adjunct therapy or adjunctive therapy) todestroy residual tumor cells that may be present after the known tumoris removed by the initial therapy (e.g. surgery), thereby preventingpossible cancer reoccurrence; neoadjuvant therapy given prior to thesurgical procedure to shrink the cancer; induction therapy to cause aremission, typically for acute leukemia; consolidation therapy (alsocalled intensification therapy) given once a remission is achieved tosustain the remission; maintenance therapy given in lower or lessfrequent doses to assist in prolonging a remission; first line therapy(also called standard therapy); second (or 3rd, 4th, etc.) line therapy(also called salvage therapy) is given if a disease has not responded orreoccurred after first line therapy; and palliative therapy (also calledsupportive therapy) to address symptom management without expecting tosignificantly reduce the cancer.

The terms “preventing” and “prevention” refer to the administration ofan agent or composition to a clinically asymptomatic individual who issusceptible or predisposed to a particular adverse condition, disorder,or disease, and thus relates to the prevention of the occurrence ofsymptoms and/or their underlying cause.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Certain methodologies of the instant invention include a step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isa control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing a treatment and/or agentadministration methodology, as described herein. For example, atranscription rate, mRNA level, translation rate, protein level,biological activity, cellular characteristic or property, genotype,phenotype, etc. can be determined prior to introducing a treatmentand/or agent of the invention to a subject. In another embodiment, a“suitable control” or “appropriate control” is a value, level, feature,characteristic, property, etc. determined in a cell or organism, e.g., acontrol or normal cell or organism, exhibiting, for example, normaltraits. In yet another embodiment, a “suitable control” or “appropriatecontrol” is a predefined value, level, feature, characteristic,property, etc.

Genes: All genes, gene names, and gene products disclosed herein areintended to correspond to homologs from any species for which thecompositions and methods disclosed herein are applicable. Thus, theterms include, but are not limited to genes and gene products fromhumans and mice. It is understood that when a gene or gene product froma particular species is disclosed, this disclosure is intended to beexemplary only, and is not to be interpreted as a limitation unless thecontext in which it appears clearly indicates. Thus, for example, forthe genes or gene products disclosed herein, which in some embodimentsrelate to mammalian nucleic acid and amino acid sequences, are intendedto encompass homologous and/or orthologous genes and gene products fromother animals including, but not limited to other mammals, fish,amphibians, reptiles, and birds. In preferred embodiments, the genes,nucleic acid sequences, amino acid sequences, peptides, polypeptides andproteins are human.

Genbank and NCBI submissions indicated by accession number cited hereinare incorporated herein by reference.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below.

DETAILED DESCRIPTION

Embodiments of the invention are directed to inducing and/or enhancingan immune response to neoplastic diseases.

BCG therapy of bladder cancer results in extensive activation of theimmune system. Within hours of BCG instillation, a marked increase inthe number of leukocytes in the urine can be detected. These leukocytesconsist mainly of granulocytes and, to a lesser degree, of macrophagesand lymphocytes (De Boer, E, C. el al. Presence of activated lymphocytesin the urine of patients with superficial bladder cancer afterintravesical immunotherapy with Bacillus Calmette-Guerin. CancerImmunol. Immunother. 33, 411-416 (1991)). Similarly, an influx of immunecells can be found in the bladder wall after BCG therapy (Bohle A. etal., Effects of local Bacillus Calmette-Guerin therapy in patients withbladder carcinoma on immunocompetent cells of the bladder wall. J. Urol,144 53-58 (1990)). Additional evidence of immune activation is therelease of a wide variety of cytokines and chemokines into the urinefollowing BCG therapy (Redelman-Sidi G. et al, Nat Rev Urol. 2014 March;11(3):153-62). Histopathologically, post-treatment bladder biopsies inpatients treated with BCG reveal erosion of the superficial epithelium,and submucosal granulomatous inflammation, with oedema and non caseatinggranulomas surrounded by a lymphoplasmacytic and eosinophilic infiltrate(Lage, J. M. et al. Histological parameters and pitfalls in theinterpretation of bladder biopsies in Bacillus Calmette-Guerin treatmentof superficial bladder cancer. J. Urol. 135, 916-919 (1986)).

The majority of lymphocytes in the urine of patients treated with BCGare T cells, most of which are CD4⁺ (De Boer, E. C. et al. Presence ofactivated lymphocytes in the urine of patients with superficial bladdercancer after intravesical immunotherapy with Bacillus Calmette-Guerin.Cancer Immunol. Immunother. 33, 411-416 (1991)). T cells, again mostlyCD4⁺, can also be found infiltrating the bladder mucosa for months afterBCG therapy (Bohle, A. et al., J. Urol. 144, 53-58 (1990); Boccafoschi,C. et al. Immunophenotypic characterization of the bladder mucosainfiltrating lymphocytes after intravesical BCG treatment forsuperficial bladder carcinoma. Eur. Urol. 21, 304-308 (1992)). AlthoughCD4⁺ lymphocytes predominate, both CD4⁺ and CD8⁺ lymphocytes seem to berequired for effective BCG therapy.

The cytotoxicity of BCG-specific NK cells, which have also been termedBCG-activated killer (BAK) cells (Brandau, S. & Bohle, A. Activation ofnatural killer cells by Bacillus Calmette-Guerin. Eur. Urol. 39, 518-524(2001)), can be enhanced by IL-12 and interferon (IFN)-γ, and isinhibited by IL-1 (Suttmann, H. et al. Mechanisms of BacillusCalmette-Guerin mediated natural killer cell activation. J. Urol. 172,1490-1495 (2004)). Killing of bladder cancer cells by BAK cells seems toinvolve perforin, which is a cytolytic protein that is released fromgranules and forms a pore in the plasma membrane of the target cell(Brandau, S. et al. Perforin-mediated lysis of tumor cells byMycobacterium bovis Bacillus Calmette-Guerin-activated killer cells.Clin. Cancer Res. 6, 3729-3738 (2000)).

Dendritic cells have been postulated to initiate activation of T cellsafter BCG administration. Although some evidence from in vitro studiessupports this hypothesis, a role for dendritic cells in response to BCGhas not been clearly defined. Immature dendritic cells have beenidentified in the urine of patients with bladder cancer who were treatedwith BCG (Beatty, J. D. et al. Urine dendritic cells: a noninvasiveprobe for immune activity in bladder cancer? BJU Int. 94, 1377-1383(2004)), and in vitro, dendritic cells that were exposed to BCG canactivate NK cells and γδ T cells, and induce their cytotoxicity againstBCG-infected bladder cancer cells (Naoe, M. et al. BacillusCalmette-Guerin-pulsed dendritic cells stimulate natural killer T cellsand gamma delta T cells. Int. J Urol. 14, 532-538 (2007); Higuchi, T. etal. A possible mechanism of intravesical BCG therapy for human bladdercarcinoma: involvement of innate effector cells for the inhibition oftumor growth. Cancer Immunol. Immunother. 58, 1245-1255 (2009)). Similarto the finding for tumor-associated macrophages, patients with highlevels of tumor-associated dendritic cells prior to BCG treatment weremore likely to experience cancer recurrence after BCG therapy (Ayari, C.et al. Bladder tumor infiltrating mature dendritic cells and macrophagesas predictors of response to Bacillus Calmette-Guerin immunotherapy.Eur. Urol. 55, 1386-1395 (2009)). As is the case with tumor-associatedmacrophages, this finding might be explained by immunosuppressioninduced by specific subsets of tumor-associated dendritic cells(Hurwitz, A. A. & Watkins, S. K. Immune suppression in the tumormicroenvironment: a role for dendritic cell-mediated tolerization of Tcells. Cancer Immunol. Immunother. 61, 289-293 (2012)).

BCG therapy is followed by a massive release of cytokines into the urineof treated patients. These cytokines include IL-1, IL-2, IL-5, IL-6,IL-8, IL-10, IL-12, IL-18, TNF, IFN-γ, and granulocyte—macrophagecolony-stimulating factor (GM-CSF) (De Boer, E. C. et al. CancerImmunol. Immunother. 34, 306-312 (1992); Eto, M. et al. Importance ofurinary interleukin-18 in intravesical immunotherapy with BacillusCalmette-Guerin for superficial bladder tumors. Urol. Int. 75, 114-118(2005)), as well as the chemokines macrophage-derived chemokine (MDC),monocyte chemoattractant protein (MCP)-1, MIP-la, andinterferon-inducible protein (IP)-10 (Luo, Y., et al. Mycobacteriumbovis bacillus Calmette-Guerin (BCG) induces human CC- andCXC-chemokines in vitro and in vivo. Clin. Exp. Immunol. 147, 370-378(2007)). Although the array of cytokines found in the urine of patientstreated with BCG cannot be strictly categorized as corresponding to aT_(H)1 or T_(H)2 response, the presence of IL-2, IL-12, and IFN-γ, andthe absence of IL-4, are more consistent with a T_(H)1-like response.Another cytokine that has been evaluated in response to BCG is TRAIL, amember of the TNF family that is expressed by various immune cells,including cytotoxic lymphocytes, NK cells, and neutrophils.

In addition to the local inflammatory response in the bladder wall, BCGtherapy induces a systemic immune response. More than 40% of patientsreceiving intra-vesical BCG instillation experience conversion of apreviously negative tuberculin skin test (Kelley, D. R. et al.Prognostic value of purified protein derivative skin test and granulomaformation in patients treated with intravesical BacillusCalmette-Guerin. J. Urol. 135, 268-271 (1986)). Furthermore, patientstreated with BCG have increased serum levels of IL-2 and IFN-γ, andperipheral blood mononuclear cells in patients who have receivedrepeated instillations of BCG exhibit increased killing activity againstan NK-cell resistant cancer cell line, compared with before BCGtreatment (Taniguchi, K. et al. Systemic immune response afterintravesical instillation of Bacillus Calmette-Guerin (BCG) forsuperficial bladder cancer. Clin. Exp. Immunol. 115, 131-135 (1999)).

The immune response to BCG is preceded by an interaction between BCG andurothelial cells, which is essential to achieving antitumor activity.The initial step is attachment of BCG to urothelial cells. This step isfacilitated by fibronectin, a glycoprotein that is part of theextracellular matrix and that can also be found in urine in a solubleform. BCG attaches to fibronectin through its fibronectin attachmentprotein (FAP) (Zhao, W. et al. Role of a Bacillus Calmette-Guerinfibronectin attachment protein in BCG-induced antitumor activity. Int.J. Cancer 86, 83-88 (2000)). In turn, fibronectin is thought to attachto urothelial cells through integrin α5β1 (Coplen, D. E., et al.Characterization of fibronectin attachment by a human transitional cellcarcinoma line, T24. J. Urol. 145, 1312-1315 (1991)). In vitro, BCGattachment and internalization is enhanced by addition of exogenousfibronectin and inhibited by antibodies against fibronectin or integrinsα5 or β1 (Kuroda, K., et al. Characterization of the internalization ofBacillus Calmette-Guerin by human bladder tumor cells. J. Clin. Invest.91, 69-76 (1993)).

Bladder cancer cells can directly secrete immune-activating effectorsfollowing internalization of BCG. The main cytokine studied in thiscontext has been IL-6, which is released from bladder cancer cellsexposed to BCG.

Dendritic Cells

Dendritic Cells (DC) are the most powerful antigen presenting cells ofthe immune system, capable of stimulating naïve and memory CD8⁺ T-cellsas well as B-cells and CD4⁺ helper T-cells. In the immature state DC arepresent in blood and tissues, processing foreign antigens forpresentation to the immune system. The uptake of presentable antigenstimulates maturation of DC and promotes DC migration to lymph nodes,where these cells can directly interact with immune effector cells.Mature DC are capable of stimulating T helper type-1 immune responsesand antigen specific CD8⁺ cytotoxic T-lymphocytes (CTL), but within thetumor microenvironment DC promote tumor tolerance, facilitating T helpertype-2 responses. Therefore DC can exert both strong positive andnegative influences on the acquisition of tumor specific cellular immuneresponses.

DC vaccines have generally consisted of autologous monocytes that arematured in vitro and pulsed with antigen before injection. Each step ofDC vaccine production, DC generation, antigen loading, in vitromaturation, and inoculation with or without adjuvant is an opportunityto enhance efficacy. DC vaccine research has therefore focused onexpanding the available sources of DC and improving DC immunogenicity,optimizing the source and presentation of antigen, developing new immuneadjuvants, and investigation of concomitant immunomodulation orchemotherapy. (Kalijn F. Bol et al. “Dendritic Cell—Based Immunotherapy:State of the Art and Beyond.” Clinical Cancer Research, 2016;22:1897-1906; Elster, Jennifer D et al. “Dendritic cell vaccines: Areview of recent developments and their potential pediatricapplication.” Human Vaccines & Immunotherapeutics vol. 12, 9 ( ):2232-9. doi:10.1080/21645515.2016.1179844).

Interleukin-15

IL-15 is a pleiotropic cytokine that plays various roles in the innateand adaptive immune systems, including the development, activation,homing and survival of immune effector cells, especially NK, NK-T andCD8⁺ T cells (Cooper, M. A., et al., Blood, 2001. 97(10): p. 3146-51).IL-15, a member of the common gamma chain (γc) cytokine family, binds toa receptor complex that consists of IL-15Rα, IL-2Rβ and the γc chain(Grabstein, K. H., et al., Science, 1994. 264(5161): p. 965-8; Giri, J.G., et al., Embo J, 1995. 14(15): p. 3654-63). Furthermore, IL-15functions as a key regulator of development, homeostasis and activity ofNK cells (Prlic, M., et al., J Exp Med, 2003. 197(8): p. 967-76; Carson,W. E., et al., J Clin Invest, 1997. 99(5): p. 937-43). IL-15administration to normal mice or overexpression of IL-15 in thetransgenic mouse model increases the number and percentage of NK cellsin the spleen (Evans, R., et al., Cell Immunol, 1997. 179(1): p. 66-73;Marks-Konczalik, J., et al., Proc Natl Acad Sci USA, 2000. 97(21): p.11445-50), the proliferation and survival of NK cells, as well as theircytolytic activity and cytokine secretion. IL-15 administration couldalso increase the NK cell number and function in recipients of stem celltransplantation (Katsanis, E., et al., Transplantation, 1996. 62(6): p.872-5; Judge, A. D., et al., J Exp Med, 2002. 196(7): p. 935-46;Alpdogan, O., et al., Blood, 2005. 105(2): p. 865-73; Sauter, C. T., etal., Bone Marrow Transplantation, 2013. 48(9): p. 1237-42).

The primary limitations in clinical development of recombinant humanIL-15 (rhIL-15) are low production yields in standard mammalian cellexpression systems and a short serum half-life (Ward, A., et al.,Protein Expr Purif, 2009. 68(1): p. 42-8; Bessard, A., et al., MolCancer Ther, 2009. 8(9): p. 2736-45). The formation of the IL-15:IL-15Rαcomplex, with both proteins co-expressed in the same cell can stimulateimmune effector cells bearing the IL-2βγc receptor through atrans-presentation mechanism. In addition, when IL-15 is bound toIL-15Rα, it increased the affinity of the IL-15 to IL-2Rβ approximately150-fold, when compared with free IL-15 (Ring, A. M., et al., NatImmunol, 2012. 13(12): p. 1187-95). A superagonist mutant of IL-15(IL-15N72D), which has increased IL-2Rβ binding ability (4-5 fold higherthan native IL-15) has been identified for therapeutic usages (Zhu, X.,et al., Novel human interleukin-15 agonists. J Immunol, 2009. 183(6): p.3598-607). The strong interaction of IL-15N72D and soluble IL-15Rα wasexploited to create an IL-15 superagonist complex with IL-15N72D boundto IL-15RαSu/Fc. The soluble fusion protein, IL-15RαSu/Fc, was createdby linking the human IL-15RαSu domain with human IgG1 containing the Fcdomain. Studies on IL-15:IL-15Rα complexes show an advantage ofincreased intracellular stability of IL-15 (Bergamaschi, C., et al., JBiol Chem, 2008. 283(7): p. 4189-99; Duitman, E. H., et al., Mol CellBiol, 2008. 28(15): p. 4851-61). Co-expression of both the IL-15N72D andIL-15RαSu/Fc proteins resulted in a soluble and stable complex withsignificantly longer serum half-life and increased biological activity,compared to native IL-15 (Han, K. P., et al., Cytokine, 2011. 56(3): p.804-10). As indicated above, this IL-15N72D:IL-15RαSu/Fc complex (N-803)was >10-fold more active than free IL-15 in promoting in vitroproliferation of IL-15-dependent cells (Zhu, X., et al., Novel humaninterleukin-15 agonists. J Immunol, 2009. 183(6): p. 3598-607). N-803has potent anti-tumor activity in syngeneic murine models of multiplemyeloma (Xu, W., et al., Cancer Res, 2013. 73(10): p. 3075-86).

IL-15:IL-15Rα Complex

As defined above, an IL-15:IL-15Rα fusion protein complex can refer to acomplex having IL-15 non-covalently bound to the soluble IL-15Rα domainof the native IL-15Rα. In some cases, the soluble IL-15Rα is covalentlylinked to a biologically active polypeptide and/or to an IgG Fc domain.The IL-15 can be either IL-15 or IL-15 covalently linked to a secondbiologically active polypeptide. The crystal structure of theIL-15:IL-15Rα complex is shown in Chirifu et al., 2007 Nat Immunol 8,1001-1007, incorporated herein by reference.

In certain embodiments, the IL-15Rα comprises IL-15RαSushi (IL-15RαSu).In other embodiments, the IL-15 is a variant IL-15 (e.g., IL-15N72D).

In certain embodiments of the soluble fusion protein complexes of theinvention, the IL-15 polypeptide is an IL-15 variant having a differentamino acid sequence than native IL-15 polypeptide. The human IL-15polypeptide is referred to herein as huIL-15, hIL-15, huIL15, hIL15,IL-15 wild type (wt) and variants thereof are referred to using thenative amino acid, its position in the mature sequence and the variantamino acid. For example, huIL15N72D refers to human IL-15 comprising asubstitution of N to D at position 72. In certain embodiments, the IL-15variant functions as an IL-15 agonist as demonstrated, e.g., byincreased binding activity for the IL-15RβγC receptors compared to thenative IL-15 polypeptide. In certain embodiments, the IL-15 variantfunctions as an IL-15 antagonist as demonstrated by e.g., decreasedbinding activity for the IL-15RβγC receptors compared to the nativeIL-15 polypeptide. In certain embodiments, the IL-15 variant hasincreased binding affinity or a decreased binding activity for theIL-15RβγC receptors compared to the native IL-15 polypeptide. In certainembodiments, the sequence of the IL-15 variant has at least one (i.e.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid change compared tothe native IL-15 sequence. The amino acid change can include one or moreof an amino acid substitution or deletion in the domain of IL-15 thatinteracts with IL-15Rβ and/or IL-15RγC. In certain embodiments, theamino acid change is one or more amino acid substitutions or deletionsat position 8, 61, 65, 72, 92, 101, 108, or 111 of the mature humanIL-15 sequence. For example, the amino acid change is the substitutionof D to N or A at position 8, D to A at position 61, N to A at position65, N to R at position 72 or Q to A at position 108 of the mature humanIL-15 sequence, or any combination of these substitutions. In certainembodiments, the amino acid change is the substitution of N to D atposition 72 of the mature human IL-15 sequence.

N-803

N-803 comprises an IL-15 mutant with increased ability to bind IL-2Rβγand enhanced biological activity (U.S. Pat. No. 8,507,222, incorporatedherein by reference). This super-agonist mutant of IL-15 was describedin a publication (J. Immunol 2009 183:3598) and a patent has been issuedby the U.S. Patent & Trademark Office on the super agonist and severalpatents applications are pending (e.g., U.S. Ser. Nos. 12/151,980 and13/238,925). This IL-15 super-agonist in combination with a solubleIL-15a receptor fusion protein (IL-15RαSu/Fc) results in a proteincomplex with highly potent IL-15 activity in vitro and in vivo (Han etal., 2011, Cytokine, 56: 804-810; Xu, et al., 2013 Cancer Res.73:3075-86, Wong, et al., 2013, Oncolmmunology 2:e26442). This IL-15super agonist complex (IL-15N72D:IL-15RαSu/Fc) is referred to as N-803.Pharmacokinetic analysis indicated that the complex has a half-life of25 hours following i.v. administration in mice. N-803 exhibitsimpressive anti-tumor activity against aggressive solid andhematological tumor models in immunocompetent mice. It can beadministered as a monotherapy using a twice weekly or weekly i.v. doseregimen or as combinatorial therapy with an antibody. The N-803anti-tumor response is also durable. Tumor-bearing mice that were curedafter N-803 treatment were also highly resistant to re-challenge withthe same tumor cells indicating that N-803 induces effectiveimmunological memory responses against the re-introduced tumor cells.

Fc Domain

N-803 comprises an IL-15N72D:IL-15RαSu/Fc fusion complex. Fusionproteins that combine the Fc regions of IgG with the domains of anotherprotein, such as various cytokines and soluble receptors have beenreported (see, for example, Capon et al., Nature, 337:525-531, 1989;Chamow et al., Trends Biotechnol., 14:52-60, 1996; U.S. Pat. Nos.5,116,964 and 5,541,087). The prototype fusion protein is a homodimericprotein linked through cysteine residues in the hinge region of IgG Fc,resulting in a molecule similar to an IgG molecule without the heavychain variable and C_(H1) domains and light chains. The dimeric natureof fusion proteins comprising the Fc domain may be advantageous inproviding higher order interactions (i.e. bivalent or bispecificbinding) with other molecules. Due to the structural homology, Fc fusionproteins exhibit an in vivo pharmacokinetic profile comparable to thatof human IgG with a similar isotype. Immunoglobulins of the IgG classare among the most abundant proteins in human blood, and theircirculation half-lives can reach as long as 21 days. To extend thecirculating half-life of IL-15 or an IL-15 fusion protein and/or toincrease its biological activity, fusion protein complexes containingthe IL-15 domain non-covalently bound to IL-15RαSu covalently linked tothe Fc portion of the human heavy chain IgG protein have been made(e.g., N-803).

The term “Fe” refers to a non-antigen-binding fragment of an antibody.Such an “Fe” can be in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulins, although IgG 1 and IgG2 arepreferred. Native Fc's are made up of monomeric polypeptides that may belinked into dimeric or multimeric forms by covalent (i.e., disulfidebonds) and non-covalent association. The number of intermoleculardisulfide bonds between monomeric subunits of native Fc molecules rangesfrom 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is adisulfide-bonded dimer resulting from papain digestion of an IgG (seeEllison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “nativeFc” as used herein is generic to the monomeric, dimeric, and multimericforms. Fc domains containing binding sites for Protein A, Protein G,various Fc receptors and complement proteins.

In some embodiments, the term “Fc variant” refers to a molecule orsequence that is modified from a native Fc, but still comprises abinding site for the salvage receptor, FcRn. International applicationsWO 97/34631 (published Sep. 25, 1997) and WO 96/32478 describe exemplaryFc variants, as well as interaction with the salvage receptor, and arehereby incorporated by reference. Thus, the term “Fc variant” comprisesa molecule or sequence that is humanized from a non-human native Fc.Furthermore, a native Fc comprises sites that may be removed becausethey provide structural features or biological activity that are notrequired for the fusion molecules of the present invention. Thus, incertain embodiments, the term “Fc variant” comprises a molecule orsequence that lacks one or more native Fc sites or residues that affector are involved in (1) disulfide bond formation, (2) incompatibilitywith a selected host cell (3) N-terminal heterogeneity upon expressionin a selected host cell, (4) glycosylation, (5) interaction withcomplement, (6) binding to an Fc receptor other than a salvage receptor,(7) antibody-dependent cell-mediated cytotoxicity (ADCC), or (8)antibody dependent cellular phagocytosis (ADCP). Fc variants aredescribed in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by recombinant geneexpression or by other means.

Linkers

In some cases, the fusion protein complexes of the invention alsoinclude a flexible linker sequence interposed between the IL-15 orIL-15Rα domains. The linker sequence should allow effective positioningof the polypeptide with respect to the IL-15 or IL-15Rα domains to allowfunctional activity of both domains.

In certain cases, the soluble fusion protein complex has a linkerwherein the first polypeptide is covalently linked to IL-15 (orfunctional fragment thereof) by a polypeptide linker sequence. In otheraspects, the soluble fusion protein complex as described herein has alinker wherein the second polypeptide is covalently linked to IL-15Rαpolypeptide (or functional fragment thereof) by polypeptide linkersequence.

The linker sequence is preferably encoded by a nucleotide sequenceresulting in a peptide that can effectively position the binding grooveof a TCR molecule for recognition of a presenting antigen or the bindingdomain of an antibody molecule for recognition of an antigen. As usedherein, the phrase “effective positioning of the biologically activepolypeptide with respect to the IL-15 or IL-15Rα domains”, or othersimilar phrase, is intended to mean the biologically active polypeptidelinked to the IL-15 or IL-15Rα domains is positioned so that the IL-15or IL-15Rα domains are capable of interacting with each other to form aprotein complex. For example, the IL-15 or IL-15Rα domains areeffectively positioned to allow interactions with immune cells toinitiate or inhibit an immune reaction, or to inhibit or stimulate celldevelopment.

The fusion protein complexes of the invention preferably also include aflexible linker sequence interposed between the IL-15 or IL-15Rα domainsand the immunoglobulin Fc domain. The linker sequence should alloweffective positioning of the Fc domain, biologically active polypeptideand IL-15 or IL-15Rα domains to allow functional activity of eachdomain. For example, the Fc domains are effectively positioned to allowproper fusion protein complex formation and/or interactions with Fcreceptors on immune cells or proteins of the complement system tostimulate Fc-mediated effects including opsonization, cell lysis,degranulation of mast cells, basophils, and eosinophils, and other Fcreceptor-dependent processes; activation of the complement pathway; andenhanced in vivo half-life of the fusion protein complex.

Linker sequences can also be used to link two or more polypeptides ofthe biologically active polypeptide to generate a single-chain moleculewith the desired functional activity.

Preferably, the linker sequence comprises from about 7 to 20 aminoacids, more preferably from about 10 to 20 amino acids. The linkersequence is preferably flexible so as not hold the biologically activepolypeptide or effector molecule in a single undesired conformation. Thelinker sequence can be used, e.g., to space the recognition site fromthe fused molecule. Specifically, the peptide linker sequence can bepositioned between the biologically active polypeptide and the effectormolecule, e.g., to chemically cross-link same and to provide molecularflexibility. The linker preferably predominantly comprises amino acidswith small side chains, such as glycine, alanine and serine, to providefor flexibility. Preferably, about 80 or 90 percent or greater of thelinker sequence comprises glycine, alanine or serine residues,particularly glycine and serine residues.

Different linker sequences could be used including any of a number offlexible linker designs that have been used successfully to joinantibody variable regions together (see, Whitlow, M. et al., (1991)Methods: A Companion to Methods in Enzymology, 2:97-105).

Fusions Protein Complexes

The invention provides N-803, which is a protein complex betweenIL-15N72D and IL-15RαSu/Fc. An exemplary IL-15N72D nucleic acid sequenceis provided below (with leader peptide) (SEQ ID NO: 1):

(Leader peptide) atggagacagacacactcctgttatgggtactgctgctctgggttccaggttccaccggt- (IL-15N72D)aactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgetttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacgacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct (Stop codon) taa

An exemplary IL-15N72D amino acid sequence is provided below (withleader peptide) (SEQ ID NO: 2):

(Leader peptide) METDTLLLWVLLLWVPGSTG- (IL-15N72D)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the mature IL-15N72Dpolypeptide (SEQ ID NO: 3):

(IL-15N72D) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

An exemplary IL-15RαSu/Fc nucleic acid sequence (with leader peptide) isprovided below (SEQ ID NO: 4):

(Leader peptide) atggacagacttacttcttcattcctgctcctgattgtccctgcgtacgtcttgtcc- (IL-15RαSu) atcacgtgccctccccccatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccagggagcggtacatttgtaactctggtttcaagcgtaaagccggcacgtccagcctgacggagtgcgtgttgaacaaggccacgaatgtcgcccactggacaacccccagtctcaaatgtattaga-(IgG1 CH2-CH3 (Fc domain))gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa- (Stop codon) taa

An exemplary IL-15RαSu/Fc amino acid sequence (with leader peptide) isprovided below (SEQ ID NO: 5):

(Leader peptide) MDRLTSSFLLLIVPAYVLS- (IL-15RαSu)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR-(IgG1 CH2-CH3 (Fc domain))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some cases, the mature IL-15RαSu/Fc protein lacks the leader sequence(SEQ ID NO: 6):

(IL-15RαSu) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR- (IgG1 CH2-CH3 (Fc domain))EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Bacillus Calmette-Guerin (BCG)

Bacillus Calmette-Guérin (BCG), a live attenuated strain ofMycobacterium bovis, is currently the only agent approved by the US Foodand Drug Administration for primary therapy of carcinoma in situ (CIS)of the bladder. The original Bacillus Calmette-Guérin (BCG) strain wasdeveloped at the Pasteur Institute from an attenuated strain ofMycobacterium bovis. Two BCG products are commercially available in theUnited States. The Tice strain, which is a substrain of the originalPasteur product, is manufactured by Organon Pharmaceuticals. TheTheraCys strain is made by Aventis/Pasteur. BCG supplanted cystectomy asthe treatment of choice for CIS in the mid-1980s. BCG therapy alsoreduces the risk of recurrence, and ongoing maintenance therapy with BCGreduces the risk of progression in patients with high-grade non-muscleinvasive bladder cancer.

Bladder cancer is the only cancer in which BCG is commonly used. For BCGto be effective, all the following criteria should be met: The patientis immunocompetent, the tumor burden is small; BCG makes direct contactwith the tumor, the dose is adequate to incite a reaction.

BCG viability is an important consideration for the vaccine to beeffective. This viability is measured in colony-forming units (CFUs). Avaccine that contains no or very few live organisms would be clinicallyineffective. One dose, either an ampule or vial, may vary in weight fromone product to another, but the CFU should be similar. Tice BCG has1-8=10⁻⁸ CFUs. TheraCys has 10.5+/−8.7×10⁻⁸ CFUs.

Typically, BCG is administered in either an induction (once weekly for 6weeks) or maintenance (once weekly for 3 weeks) course. Another 6-weekcourse may be administered if a repeat cystoscopy (see image above)reveals tumor persistence or recurrence. Induction therapy combined withmaintenance therapy every 3-6 months for 1-3 years may provide morelasting results. Periodic bladder biopsies are usually necessary toassess response. Accordingly, the administration of BCG and/or N-803 canbe determined based on the progress of the patient. The guidelines fromthe American Urological Association (AUA) and the Society of UrologicOncology (SUO) (Chang S S, et al. Diagnosis and Treatment of Non-MuscleInvasive Bladder Cancer: AUA/SUO Guideline. J Urol. 2016 October 196(4):1021-9) provide further guidance on the administration of BCG.

Formulation of Pharmaceutical Compositions

The administration of compositions embodied herein, such as, immuneeffector cells, e.g. dendritic cells, BCG-primed dendritic cells,BCG-primed dendritic cells cultured with N-803, or immunotherapeuticagents e.g. N-803 and/or BCG for the treatment of a neoplasia may be byany suitable means that results in a concentration of the therapeuticthat, combined with other components, is effective in ameliorating,reducing, or stabilizing a neoplasia, e.g. bladder cancer. The N-803 andBCG may be contained in any appropriate amount in any suitable carriersubstance, and is generally present in an amount of 1-95% by weight ofthe total weight of the composition. The composition may be provided ina dosage form that is suitable for parenteral (e.g., subcutaneously,intravenously, intramuscularly, intravesicularly or intraperitoneally)administration route. The pharmaceutical compositions may be formulatedaccording to conventional pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2000 and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York).

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice or nonhuman primates, as a skilledartisan recognizes it is routine in the art to modify the dosage forhumans compared to animal models. In certain embodiments it isenvisioned that the dosage may vary from between about 0.1 μgcompound/kg body weight to about 5000 μg compound/kg body weight; orfrom about 1 μg/kg body weight to about 4000 μg/kg body weight or fromabout 10 μg/kg body weight to about 3000 μg/kg body weight. In otherembodiments this dose may be about 0.1, 0.3, 0.5, 1, 3, 5, 10, 25, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, or 5000 μg/kg body weight. In other embodiments, it is envisagedthat doses may be in the range of about 0.5 μg compound/kg body weightto about 20 μg compound/kg body weight. In other embodiments the dosesmay be about 0.5, 1, 3, 6, 10, or 20 mg/kg body weight. Of course, thisdosage amount may be adjusted upward or downward, as is routinely donein such treatment protocols, depending on the results of the initialclinical trials and the needs of a particular patient.

In particular embodiments, N-803 are formulated in an excipient suitablefor parenteral or intravesical administration. In particularembodiments, N-803 is administered at 0.5 μg/kg-about 15 μg/kg (e.g.,0.5, 1, 3, 5, 10, or 15 μg/kg).

For the treatment of bladder cancer, N-803 is administered byinstillation into the bladder. Methods of instillation are known. See,for example, Lawrencia, et al., Gene Ther 8, 760-8 (2001); Nogawa, etal., J Clin Invest 115, 978-85 (2005); Ng, et al., Methods Enzymol 391,304-13 2005; Tyagi, et al., J Urol 171, 483-9 (2004); Trevisani, et al.,J Pharmacol Exp Ther 309, 1167-73 (2004); Trevisani, et al., NatNeurosci 5, 546-51 (2002)). In certain embodiments, it is envisionedthat the N-803 dosage for instillation may vary from between about 5 and1000 μg/dose. In other embodiments the intravesical doses may be about25, 50, 100, 200, or 400 μg/dose. In other embodiments, N-803 isadministered by instillation into the bladder in combination withstandard therapies, including mitomycin C or BCG.

Pharmaceutical compositions are formulated with appropriate excipientsinto a pharmaceutical composition that, upon administration, releasesthe therapeutic in a controlled manner. Examples include single ormultiple unit tablet or capsule compositions, oil solutions,suspensions, emulsions, microcapsules, microspheres, molecularcomplexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical compositions embodied herein, such as, immuneeffector cells, e.g. dendritic cells, BCG-primed dendritic cells,BCG-primed dendritic cells cultured with N-803, or immunotherapeuticagents e.g. N-803 and/or BCG may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intravesicularly, intraperitoneal, or the like) in dosageforms, formulations, or via suitable delivery devices or implantscontaining conventional, non-toxic pharmaceutically acceptable carriersand adjuvants. The formulation and preparation of such compositions arewell known to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions embodied herein, such as, immune effector cells, e.g.dendritic cells, BCG-primed dendritic cells, BCG-primed dendritic cellscultured with N-803, or immunotherapeutic agents e.g. N-803 and/or BCGfor intravesical or parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules, syringes or bags), or in vialscontaining several doses and in which a suitable preservative may beadded (see below). The composition may be in the form of a solution, asuspension, an emulsion, an infusion device, or a delivery device forimplantation, or it may be presented as a dry powder to be reconstitutedwith water or another suitable vehicle before use. Apart from the activeagent that reduces or ameliorates a neoplasia, the composition mayinclude suitable parenterally acceptable carriers and/or excipients. Theactive therapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions comprising N-803and/or BCG may be in a form suitable for sterile injection. To preparesuch a composition, the suitable active antineoplastic therapeutic(s)are dissolved or suspended in a parenterally acceptable liquid vehicle.Among acceptable vehicles and solvents that may be employed are water,water adjusted to a suitable pH by addition of an appropriate amount ofhydrochloric acid, sodium hydroxide or a suitable buffer,1,3-butanediol, Ringer's solution, and isotonic sodium chloride solutionand dextrose solution. The aqueous formulation may also contain one ormore preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).In cases where one of the compounds is only sparingly or slightlysoluble in water, a dissolution enhancing or solubilizing agent can beadded, or the solvent may include 10-60% w/w of propylene glycol or thelike.

The present invention provides methods of treating neoplastic and/ordisorders or symptoms thereof which comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising a composition embodied herein, such as, immune effectorcells, e.g. dendritic cells, BCG-primed dendritic cells, BCG-primeddendritic cells cultured with N-803, or immunotherapeutic agents e.g.N-803 and/or BCG herein to a subject (e.g., a mammal such as a human).Thus, one embodiment is a method of treating a subject suffering from orsusceptible to a neoplastic or disorder or symptom thereof. The methodincludes the step of administering to the mammal a therapeutic amount ofan amount of a composition embodied herein sufficient to treat thedisease or disorder or symptom thereof, under conditions such that thedisease or disorder is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a neoplastic disease, disorder,or symptom thereof. Determination of those subjects “at risk” can bemade by any objective or subjective determination by a diagnostic testor opinion of a subject or health care provider (e.g., genetic test,enzyme or protein marker, Marker (as defined herein), family history,and the like). N-803 may be used in the treatment of any other disordersin which an increase in an immune response is desired.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof associated withneoplasia in which the subject has been administered a therapeuticamount of a compound herein sufficient to treat the disease or symptomsthereof. The level of Marker determined in the method can be compared toknown levels of Marker in either healthy normal controls or in otherafflicted patients to establish the subject's disease status. Inpreferred embodiments, a second level of Marker in the subject isdetermined at a time point later than the determination of the firstlevel, and the two levels are compared to monitor the course of diseaseor the efficacy of the therapy. In certain preferred embodiments, apre-treatment level of Marker in the subject is determined prior tobeginning treatment according to this invention; this pre-treatmentlevel of Marker can then be compared to the level of Marker in thesubject after the treatment commences, to determine the efficacy of thetreatment.

Combination Therapies

The compositions embodied herein, such as, immune effector cells, e.g.dendritic cells, BCG-primed dendritic cells, BCG-primed dendritic cellscultured with N-803, or immunotherapeutic agents e.g. N-803 and/or BCG,can be administered in combination with an anti-neoplasia such as achemotherapeutic agent, e.g. mitomycin C, an antibody, e.g., atumor-specific antibody or an immune-checkpoint inhibitor. Thecompositions may be administered simultaneously or sequentially. In someembodiments, the chemotherapeutic treatment is an established therapyfor the disease indication and addition of, for example, dendritic cellsisolated from a subject, the treatment improves the therapeutic benefitto the patients. Such improvement could be measured as increasedresponses on a per patient basis or increased responses in the patientpopulation. Combination therapy could also provide improved responses atlower or less frequent doses of chemotherapeutic agent resulting in abetter tolerated treatment regimen.

If desired, the immune effector cells, e.g. dendritic cells obtainedfrom subjects having been administered BCG and cultured with N-803, areadministered in combination with any conventional therapy, including butnot limited to, surgery, radiation therapy, chemotherapy, protein-basedtherapy or biological therapy. Chemotherapeutic drugs include alkylatingagents (e.g., platinum-based drugs, tetrazines, aziridines,nitrosoureas, nitrogen mustards), anti-metabolites (e.g., anti-folates,fluoropyrimidines, deoxynucleoside analogues, thiopurines),anti-microtubule agents (e.g., vinca alkaloids, taxanes), topoisomeraseinhibitors (e.g., topoisomerase I and II inhibitors), cytotoxicantibiotics (e.g., anthracyclines) and immunomodulatory drugs (e.g.,thalidomide and analogs).

Anti-Cancer Therapeutic Agents

The methods of the invention may include administration of secondtherapeutic agent or treatment with a second therapy (e.g., atherapeutic agent or therapy that is standard in the art). Exemplarytherapeutic agents include chemotherapeutic agents. A “chemotherapeuticagent” is a chemical compound useful in the treatment of cancer.Examples of chemotherapeutic agents include mitomycin C, Erlotinib(TARCEVA™, Genentech/OSI Pharm.), Bortezomib (VELCADE™, MillenniumPharm.), Fulvestrant (FASLODEX™, Astrazeneca), Sutent (SU11248, Pfizer),Letrozole (FEMARA′, Novartis), Imatinib mesylate (GLEEVEC™, Novartis),PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin™, Sanofi), 5-FU(5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE™, Wyeth),Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336),Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA™,Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such asThiotepa and CYTOXAN™ cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozcicsin, carzcicsin and bizcicsin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin yl and calicheamicin omega 1(Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, anthramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, strcptonigrin, strcptozocin, tubcrcidin,ubenimcx, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacytidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosinc;arabinoside (“Ara-C”); cyclophosphamidc; thiotcpa; taxoids, e.g., TAXOL™paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR™ gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX™(tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON™ (toremifene); (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate),AROMASIN™ (exemestane), formestanie, fadrozole, RIVISOR™ (vorozole),FEMARA™ (letrozole), and ARIMIDEX™ (anastrozole); (iii) anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);(iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipidkinase inhibitors; (vii) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, such as, for example, PKC-alpha, Ralf andH-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g.,ANGIOZYME™ (ribozyme)) and a HER2 expression inhibitor; (ix) vaccinessuch as gene therapy vaccines, for example, ALLOVECTIN™ vaccine,LEUVECTIN™ vaccine, and VAXID™ vaccine; PROLEUKIN™ rIL-2; LURTOTECAN™topoisomerase 1 inhibitor; ABARELIX™ rmRH; (x) anti-angiogenic agentssuch as bevacizumab (AVASTIN™, Genentech); and (xi) pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, applicants donot admit any particular reference is “prior art” to their invention.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-18. (canceled)
 19. A vaccine composition comprising Bacillus Calmette-Guerin (BCG) primed dendritic cells and an IL-15:IL-15Rα fusion protein complex.
 20. The vaccine composition of claim 19 wherein the IL 15:IL-15Rα complex comprises an IL-15N72D:IL-15RαSu/Fc complex.
 21. The vaccine composition of claim 20, wherein the IL-15N72D:IL-15RαSu/Fc complex comprises a dimeric IL-15RαSu/Fc and two IL-15N72D molecules.
 22. The vaccine composition of claim 19, wherein the dendritic cells are isolated from a urine sample of a subject diagnosed with urothelial/bladder carcinoma.
 23. The vaccine composition of claim 19, further comprising an adjuvant.
 24. The vaccine composition of claim 19, wherein the composition further comprises urothelial/bladder carcinoma antigens.
 25. The vaccine composition of claim 20, wherein the IL-15N72D:IL-15RαSu/Fc complex is N-803.
 26. The vaccine composition of claim 19, wherein the vaccine is formulated in an administration mode selected from the group consisting of systemic, intravenous, local, subcutaneous, intramuscular, inhalation and intraperitoneal. 